For many years, West Nile virus (WN) has been recognized as one of the most widely distributed flavivirus with geographic range including Africa, the Middle East, Europe and Australia. During 1999, WN virus first established itself in the USA, initially in the Northeast. The virus then spread rapidly throughout North America and is likely to extend its range throughout the hemisphere. Because of this unexpected WN spread, this virus is considered a significant re-emerging disease threat and it has become a public health problem in the USA. Currently, a licensed human vaccine is not available to prevent WN virus disease. Mosquito control is the only practical strategy to combat the spread of disease; however, effective spraying in an attempt to eliminate mosquitoes is difficult to perform in urban areas. Clearly, an effective vaccine is needed to protect at-risk populations, especially since the magnitude of epidemic disease continues to increase. For these reasons, we are interested in developing a vaccine against WN. We constructed viable WN/DEN4 chimeras in which the structural preM and E protein genes of the WN virus were substituted for the corresponding genes of dengue type 4 virus (DEN4). We also generated a WN/DEN4 chimera with a 30-nucleotide deletion in the 3'-noncoding region. Both chimeric (WN/DEN4 and its mutant) and parental (WN strain NY99 and DEN4 strain 814669) viruses were evaluated in mice, birds, horses, mosquitoes, and monkeys. Both chimeric WN/DEN4 and WN/DEN4-d30 virus were more than 10,000 times less neurovirulent in suckling mice inoculated IC than its WN parent. Peripheral virulence of chimeric and parental viruses was evaluated in 3-week-old Swiss and SCID mice inoculated IP with decimal dilutions of virus ranging from 0.001 to 100,000 FFU. Both chimeras were highly attenuated compared to their WN parent. Notably, IP inoculation of 10,000 FFU of the deletion mutant chimera or 100,000 FFU of the unmodified chimera did not induce fatal encephalitis in 3-week-old Swiss mice, whereas the IP LD50 for the WN parent was 10 FFU. Also, despite the high peripheral virulence of wild-type WN strain NY99 for SCID mice (IP LD50 of 6 FFU), chimerization of WN with DEN4 completely ablated this property of its WN parent for immunodeficient mice. Thus, 3-week-old Swiss or SCID mice survived IP inoculation of 10,000 or 100,000 FFU of chimeric virus. Our observations are consistent with earlier findings that a similar large reduction of peripheral neurovirulence of TBEV or LGT occurs following chimerization with DEN4. Although highly attenuated, the WN/DEN4 chimeras stimulated a moderate to high level of serum neutralizing antibodies against WN virus. There was a strong correlation between the level of neutralizing antibodies to WN induced by immunization and resistance to subsequent lethal WN challenge. However, 100% protection against WN challenge was achieved when a single 10,000 FFU dose of WN/DEN4 chimera or its 3'-deletion mutant was used for immunization. These two molecularly engineered, live-attenuated chimeric virus vaccine candidates were also highly attenuated and protective in rhesus monkeys. Viremia in WN/DEN4-infected monkeys was reduced 100-fold compared to that in WN- or DEN4-infected monkeys. WN/DEN4 deletion mutant did not cause detectable viremia, indicating that it is even more attenuated for monkeys. These findings indicate that chimerization itself and the presence of the deletion mutation independently contribute to the attenuation phenotype for nonhuman promates. Despite their high level of attenuation in monkeys, the chimeras induced a moderate-to-high titer of neutralizing antibodies and prevented viremia in monkeys challenged with WN. Thus, the WN preM and E proteins of the chimeric viruses represent effective antigens able to induce complete protection of mice and monkeys to challenge with highly virulent WN. Our observations concerning safety, immunogenicity, and protective efficacy of the chimeric WN/DEN4 viruses in monkeys identify the WN/DEN4-d30 chimera as a promising live-attenuated virus vaccine for humans, and we plan to initiate clinical trials in early 2004.